CA2173456A1 - Inhibitors of phosphoinositide-specific phospholipase c - Google Patents

Abstract

The present invention is directed to novel .alpha.-hydroxy-phosphonate compounds which inhibit mammalian phosphoinositide-specific phospholipase-C. The compounds are potent inflammatory and analgesic agents and may be useful for the treatment of cancer.

Description

WO 95/10286 ~ t 3 9 ~ k PCT/US9~/11497 , ~

TITLE OF THE INVENTION
INHIBITORS OF PHOSPHO~OSITIDE-SPECIF~C
PHOSPHOLIPASE C

BACKGROUND OF THE INVENTION
Phospholipases C (EC 3.1.4.3) are a family of enzymes which hydrolyze the sn-3 phosphodiester bond in membrane phospholipids producing diacylglycerol and a phosphorylated polar head group. I~mm~ n phospholipase C (PLC) enzymes exhibit specificity for ~e polar head group which is hydrolyzed, i.e., phosphatidylcholine, phosphatidylinositol, etc. Recently, much interest has been generated in those PLC enzymes which selectively hydrolyze phosphoinositide lipids in response to receptor occupancy by agonist. Hydrolysis of phosphatidylinositol 4,5-bisphosphate generates two second messenger molecules; diacylglycerol, a co-factor required for activation of protein kinase C, and inositol 1,4,5-trisphosphate, a soluble second messenger molecule which promotes the release of intracellular nonmitochondrial stores of calcium (Berridge, Ann. Rev. Biochem., 56:159-193, 1987). The diacylglycerol released may be further metabolized to free arachidonic acid by sequential actions of diglycerol lipase and monoglycerol lipase.
Thus, phospholipases C are not only important enzymes in the generation of second messenger molecules, but may serve an important role in making arachidonic acid available for eicosanoid biosynthesis in select tissues.
~mm~ n tissues contain multiple distinct forms of phosphoinositide-specific PLC (Crooke and Bennett, Cell Calcium, 10:309-323, 1989; Rhee et al., Science, 244:546-550, 1989). It is proposed that each of the enzymes couple to distinct classes of cell surface receptors, i.e., PLC-~ couples to thromboxane A2, bradykinin, angiotensin and muscarinic receptors via Gqa or G11o~ (Shenker et al., J.
30 Biol. Chem., 266:9309-9312 (1991); Gutowski et al., J. Biol. Chem., 266:20519-20524 (1991); Berstein et al., J. Biol. Chem., 267:8081-8088 (1992)), PLC- y couples to growth factor receptors, etc. (Aiyar et al., Biochem. J., 261:63-70,1989; Crooke and Bennett, Cell Calcium, WO 95/10286 - PCT/US9~/11497 ~ -' 2 -10:309-323, 1989; Margolis et al., Cell, 57: 1101 - 1107, 1989; Wahl et al., Proc. Natl. Acad. Sci. USA, 86:1568-1572, 1989). Alignment of sequences from all groups reveals that the most conserved residues are clustered into two distinct regions (one of ~170 amino acids and the other of ~260 amino acids), designated the X and Y regions, respectively.
5 PLC~1 also contains src-homology regions (SH2 and SH3) that appear to mediate the interaction between the enzyme and receptors with tyrosine kinase activity, such as the epidermal growth factor (EGF) receptor (Stahl et al., Nature, 332:269-272 (1988); Katan et al., Cell, 54:171-177 (1988)).
The PLC isozymes are activated by different mech~nism~ in response to stim~ tion of specific cell surface receptors. Coupling of PLC~ to specif1c receptors or downstream effectors has not been reported but this isozyme may be associated with mech~ni~m~ that regulate the tone of vascular smooth muscle. Activation of PLC~ is achieved by l 5 guanine nucleotide binding proteins of the Gq class.
To date, the cDNA for 6 distinct PI-PLC enzymes have been cloned. The enzymes range in size from 504 amino acids to 1250 amino acids, and are remarkably divergent considering that they exhibit similar biochemical properties. 4 of the 5 enzymes (PLC-,~, PLc-~l~ PLc-~2 20 and PLC-~1) contain two domains approximately 250 amino acids in length which exhibit between 50 to 80% sequence similarity. The marked differences in DNA sequences for the different PI-PLC enzyme allows the selective targeting of one PI-PLC enzyme, without affecting other enzymes using antisense technology. The hllm~n cDNA clone has 25 been reported for PLC-~2, (Ohta et al., FEBS Lett., 242:31-35, 1988) and PLC-~yl (Burgess et al., Mol.Cell.Biol., 10:4770-4777 (1990)). The rest are rat cDNA clones. The genomic clones have not been reported for any of the PI-PLC enzymes.
All m~mm~ n tissues which have been studied exhibit one 3 or more PI-PLC enzymes. Generally, more than one enzyme exists in a single m~mm~ n cell type. PI-PLC enzymes do exhibit tissue selectivity in their distribution. PLC-,~ is found predomin~ntly in neural tissues and is the major enzyme in the brain. PLC-~1 is found in brain WO 95/10286 1 2i i~4 5~ PCTIUS9~/11497 and many peripheral tissues. PLC-o2 is found in immune cells, and PLC-o1 appears to be predomin~ntly in peripheral tissues. To date, a PI-PLC
enzyme found exclusively in infl~mm~tory cells has not been reported.
Point mutations of PLC-ol have been identified in the spontaneously hypertensive rat genome (Yagisawa et al., J. Hypertens.
9:997-1004 (1991)). Biochemical studies have demonstrated the activation of PLC-ol (5 fold) in spontaneously hypertensive rats (Kato et al., J. Biol. Chem. 267: 6483-6487 (1992)). The point mutations, situated in the putative catalytic domain, may be a major cause of the hypertension related phenomena of abnormal calcium homeostasis, a direct result of PLC-ol activation.
PI-PLC-o2 appears to be an important enzyme in imm~lnocompetent cells (Emori et al., J. Biol. Chem., 264:21885-21890).
The protein is a moderately abundant protein comprising 0.1 to 0.05% of total cytosolic protein. No information is available concerning the genetic regulation of PI-PLC enzymes, mRNA or protein stability.
It has been established that a rapid synthesis of prost~gl~n~lins (PG) from arachidonic acid in macrophages usually accompanies infl~mm~tory stimuli. Thus, inhibition of the release of arachidonic acid from macrophages would provide an effective control of PG synthesis and thereby infl~mm~tory conditions. Recently, phospholiphase C has been characterized as an enzyme which is involved in the biosynthetic phosphatidylinositol-arachidonic acid-prost~gl~ndin pathway. This finding is further subst~nti~ted by the observation that phospholipase C is inhibited by phenot~ 7ine, a compound known to inhibit the stim~ t~d release of arachidonic acid from macrophages and prost~ ndins from platelets.
Activation of T cell antigen receptor (TCR/CD3) elicits a cascade of biochemical processes which are responsible for complex biological responses ranging from i~ n~ e response to infl~mm~tion.
3 The activation of PLC~l can also be achieved through the action of nonreceptor protein tyrosine kinases in response to certain cell surface receptors in leukocytes (TCR) (Park et al., Proc. Natl. Acad. Sci. U.S A.
88:5453-5456 (1991)). PLC~ activation also occurs upon IgM ligation WO 95110286 ~ PCT/US9`~111497 in B lymphocytes, IgE receptor (FcRI) ligation in basophilic leukemia cells and IgG receptor (Fc yRI and Fc~RlI) in monocytic cells (Liao et al., Proc. Natl. Acad. Sci. U.S~. 89:3659-3663 (1992)). Thus, inhibition of PLC~ activity, may be of therapeutic value in the treatment of infl~mm~tory conditions.
PLC~ is the only isozyme that is phosphorylated by activated tyrosine kinase growth factor receptors (Rotin et al., EMBO J., 11 :559-567 (1992); Moh~mm~di et al., Mol. Cell. Biol., 11:5068-5078 (1992);
Kim et al., Cell, 65:435-441 (1991)). Following growth factor stimulation, cytosolic PLC~ is extensively and rapidly phosphorylated in il~o (50-70% of the PLC7~ molecules are modified within 5 minutes).
This phosphorylation apparently induces the relocation of PLC y to the plasma membrane where presumably it is better able to interact with its phospholipid substrates. In vitro studies lltili7.in~; enzyme that had previously been immunoprecipitated from cells suggest that the catalytic activity of the phosphorylated form of PLC yl is increased compared to that of the unphosphorylated form, although this effect also depends on the assay conditions. These results suggest that PLC~ may be an important component of mitogenic signal transduction. Furthermore, altered PLC y activity may correlate with some disease states. For example, an increase in the concenkation of PLC~has been documented in cells derived from primary hllm~n breast carcinomas which also overexpress the EGF receptor (Arteaga et al., Proc. Natl. Acad. Sci.
U.S~., 88:10435-10439 (1991)). Thus, inhibition of PLC~yactivity, particularly of the activated form, may be of therapeutic value in the 25 treatment of breast cancer.
In addition, PLC~ has been localized in vivo (immunohistochemistry) through many layers of the epidermis from a diverse series of hyperproliferative skin conditions, such as psoriasis, seborrheic keratosies and acrochordons (Nanney et al., Cell Growth and 30 Differentiation, 3:233-239 (1992)). Thus inhibition of PLC~activity may be of therapeutic value in treating benign epidermal hyperplasia.
The recent demonstration that specific members of the Gq subfamily can activate different PLC-,~ isozymes (e.g., Gqa activates ~ WO 95/10286 PCT/US94/11497 ~ ~ ~r PLC-~1) (Smrcka ef al., Science 251 :804-807 (1991); Taylor et al., FEBS
286:214-216 (1991)) provides a connection of PLC-,~ to a number of transmembrane signal transduction pathways. NIH3T3 cells transfected with an activated mutant of Gqor~ display a fully transformed phenotype, are highly tumorigenic in athymic nude mice (Kalinec et al., Mol. Cell Biol. 12:4687-4693 (1992)) and display greatly enhanced phospholipase C (PLC-,B) activity (DeVivo et al., J. Biol. Chem. 267:18263-18266 (1992)). Other mllt~t~tions in genes for the oc subunits of some heterotrimeric G proteins (Lyons et al., Science 249:655-659 (1990);
Vallar et al., Nature 330:556-558 (1987)), have been described and are associated with certain hllm~n endocrine tumours suggesting that activated G proteins may play a role in the oncogenic process. Thus, PLC-~ in addition to PLC~may be associated with hllm~n cancer.
Accordingly, it is an object of this invention to provide specific and selective inhibitors of phospholipase C which can be potent anti-infl~mm~tory and analgesic agents useful in the treatment of infl~mm~tory conditions, including rheumatoid arthritis, emphysema, bronchial infl~mm~tion, osteoa~ iLis, spondylitis, lupus, psoriasis, acute respiratory distress syndrome, gout, fever, and pain.
It is also the object of this invention to provide inhibitors of phospholipase C which are ph~rm~ceutical agents useful in the treatment of certain forms of cancer, including breast cancer, and other hyperproliferative disease states of the epidermis.
Another object of this invention is to provide ph~rm~ceutical compositions to be used in the ~lmini~tration of the novel phospholipase C inhibitors-Still a further object of this invention is to provide a method of controlling and treating infl~mm~tion and pain by ~lmini~tering an effective amount of the compounds of the instant invention in a m~mm~ n species in need of such treatment.

WO 95/10286 PCT/US9`1/11497 ~
(.3 ~

BRlE~F DESCRIPTION OF THE FIGURES

Figure 1. Plasmid pTST-PLC4 A schematic depiction of the construction of plasmid pT5T-PLC4 is shown.

Figure 2. PLC 'n amino acid sequence and cDNA encoding sequence The nucleotide sequence which encodes PLC ~1 is shown along with the corresponding amino acids of PLC ~1 which are provided underneath the cDNA sequence. The codons at 2833-2838 have been changed from the natural AGGAGG tandem to CGGCGG. (cDNA:
SEQ.ID.NO.: l; amino acid: SEQ.ID.NO.: 2).

Figure 3. cDNA encodin~ sequence which includes the epitope tag Nucleotide sequence used for expression and purification of 15 PLC ~1 (1-3879; end indicated by "*") and the neighboring BamII
restriction site. The amino acid sequence of the longest open reading frame (1291 aa) is provided underneath the corresponding nucleotides.
(cDNA: SEQ.ID.NO.: 3; amino acid: SEQ.ID.NO.: 2).

This invention relates to novel oc--phosphonate compounds of the structural formula I:

(HO)2OP ~OH

¢~ R2 or a ph~ ceutically acceptable salt or ester thereof, wherein:

WO 95110286 ~ PCT/US9~/1 1497 2t 73is6 R1 and R2 are independently selected from:
a)hydrogen; and b) 0~ R
~ \=~

provided that if R1 is hydrogen, R2 is substituent b) and if R2 is hydrogen, Rl is substituent b); and R3,R4,RS and R6 are independently selected from the group consisting 15 of:

a) hydrogen;
b) halogen;
c) Cl-C4-alkyl;
d) Cl-C4-alkoxy; and e) hydroxy, provided that at least one of R3,R4,R5 and R6 is a substituent other than hydrogen; or 25 R3 and R4 or R4 and RS are combined to form a - CH2cH2cH2cH2 -diradical.

An embodiment of the instant invention is a compound having the formula I wherein the substitutents Rl and R2 are as described hereinabove and wherein substituents R3,R4,RS and R6 are as described hereinabove provided, however, that both R3 and R4 are not methyl if RSis hydrogen and R3 and R4 or R4 and RS are not combined to form a - CH2CH2CH2CH2 - diradical.

WO95/1028G ~ G PCTIUS91/llJ97 The aLkyl substituents recited above denote straight and branched chain hydrocarbons of the length specified such as methyl, ethyl, isopropyl, isobutyl, etc.
The aLkoxy substituent represents an alkyl group as described above attached through an oxygen bridge.
The term "halogen" represents substituents selected from iodide, chloride, bromide and fluoride.
Specific compounds of the instant invention include the compounds having the formula (HO)20P~_ OH R4 R~ R5 wherein:

2 o H H -OCH3 H H -Cl H -Cl H
-Cl H H

H H -OH
H -Cl -Cl Other compounds of the instant invention include compounds having the formula:

WO 95/10286 , ~; 21 73~5~; PCT/US94/11497 _ 9 _ (H)20P

wherein:

H -Cl H

H . -CH2CH3 H

Still other compounds of the instant invention include the 15 following compounds:

(HO)2OP)~ ~3 HO~ and (HO)2OP ~CH3 WO 95/10286 't~ ~ PCT/US9-1/11497 The novel compounds of the present invention are generally prepared by a process (illustrated in Scheme 1) comprising the treatment 5 of an appropriate halogenated benzaldehyde with a suitably substituted phenol to provide the phenoxy benzaldehyde of the Formula II. This benzaldehyde is then reacted with a suitable dialkyl phosphite which provides the compound of Formula I upon saponification of the l o phosphonate moiety.

WO 95/10286 73~5C PCT/US94/11497 O``cH OH
~ +

O~CH
~ 6 O~R5 (CH3o)2op - c -OH
,:~ R6 O~R5 (HO)2P--C_OH

3 o R3 R4 W O 95/10286 ~ PCTrUS9~/11497 The starting materials of the process described above are commercially available or known in the literature.
The compounds of this invention form salts with various inorganic and organic acids and bases which are also within the scope of the invention. Such salts include ammonium salts, aLkali metal salts like sodium and potassium salts, ~lk~line earth metal salts like the calcium and magnesium salts, salts with organic bases; e.g., dicyclohexyl~mine salts, N-methyl-D-glucamine, salts with amino acids like arginine, lysine, and the like.
The non-toxic, physiologically, acceptable salts are preferred, although other salts are also useful; e.g., in isolating or purifying the product.
The salts can be formed by conventional means such as by reacting the free acid forms of the product with one or more equivalents of the appropriate base in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is then removed in vacuo or by freeze-drying or by exch~n~ing the cations of an existing salt for another cation on a suitable ion exchange resin.
It will be further appreciated that the compounds of general Formula I in this invention may be derivatized at functional groups to 20 provide prodrug derivatives which are capable of conversion back to the parent compounds in vivo. The concept of prodrug ~lmini~tration has been extensively reviewed (e.g. A.A. Sinkula in Annual Reports in Medicinal Chemistry, Vol. 10, R.V. Heinzelman, Ed., Academic Press, New York Lond, 1975, Ch. 31, pp. 306-326), H. Ferres, Drugs of Today, 25 Vol. 19:499-538 (1983) and J. Med. Chem., 18:172 (1975).

Mode of Administration and Pharmaceutical Compositions.
Because of their ability to inhibit the enzymatic activity of the various forms of phospholipase C, the compounds of Formula I of the 3 present invention can be used to reduce infl~mm~tion and relieve pain in diseases such as rheumatoid ar~ritis, emphysema, bronchial infl~mm~tion, osteoarthritis, spondylitis, lupus, psoriasis, acute respiratory distress syndrome, gout, rheumatic fever and the like.

WO 95/10286 1 7~5~ PCT/US94/11497 : ,,, J .,, Furthermore the compounds can also be used to treat cancer, especially certain forms of cancer associated with phospholipase C, such as primary hum~n breast carcinomas.
When the compounds of formula I are utilized in vivo, such compounds can be ~1mini~tered orally, topically, parentally, by inh~l~tion spray or rectally in dosage unit formnl~tions cont~ining conventional non-toxic pharmaceutically acceptable carriers.
Accordingly, the present invention also provides pharmaceutical compositions comprising the compounds of formula I
with a ph~rm~ceutically acceptable carrier.
The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. In addition to the treatment of m~mm~ls, such as mice, rats, horses, dogs, cats, etc., the compounds of the invention are effective in the treatment of hllm~n~.
Ph~rm~ceutical compositions of this invention comprise compounds of Formula I and pharmaceutical carriers suitable for the route of ~dmini~tration. Standard methods for formul~ting pharmaceutical composition of this type may be found in Remington's Pharmaceutical Sciences, Mack Publi~hin~ Company, Easton, PA.
The ph~rm~ceutical compositions cont~ining the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
Compositions intended for oral use may be prepared according to any 25 method known to the art for the m~nuf~cture of ph~ ceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparation.
3 Formulations for oral use include tablets which contain the active ingredient in admixture with non-toxic ph~rm~ceutically acceptable excipients. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium , phosphate or sodium phosphate, gr~n~ ting and disintegrating agents, for exarnple, maize starch, or alginic acid; binding agents, for example, starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tables may be uncoated or they be coated by known techniques to delay disintegration and absorption in the 5 gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl disterate may be employed.
Formlll~tions for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calciurn carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil mediurn, for example, peanut oil, liquid paraffin, or olive oil.
A typical tablet or capsule may contain the following:
15 Ingredient Percent w/w Lactose, spray-dried 40-99 Magnesium stearate 1-2 Cornstarch 10-20 Active ingredient 0.001-20 The pha~maceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example, olive oil or arachis oils, or a mineral oil, for example, liquid paraffin or mixt~lres of those. Suitable emulsifying 2 5 agents may be naturally-occurring gums, for example, gum acacia or gum tragacanth, naturally-occurring phosphatides, for example, soybean lecithin; and esters including partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan mono-oleate, and condensation products of said partial esters with ethylene oxide, for example, 30 polyoxyethylene sorbitan mono-oleate. The emulsions may also contain sweetening and flavoring agents.
Syrups and elixirs may be form~ ted with sweetening agents, for exarnple, glycerol, sorbitol or sucrose. Such formulations may WO 95/10286 21 7 ` PCT/US9~111497 also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may be - form~ ted according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.
Parenteral compositions are prepared in conventional suspension or solution forms, as emulsions or as solid forms for reconstruction. Suitable carriers are water, saline, dextrose, Hank's solution, Ringer's solution, glycerol, and the like. Parenteral a~lmini.stration is usually by injection which may be subcutaneous, intramuscular or intravenous.
The sterile injectable prepartion may be sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, 1,3-butanediol. Ringer's solution and isotonic 15 sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids such as oleic acid also find use in the preparation of injectables.
The compounds of formula I can also be ~lmini.stered in the form of suppositories for rectal ~(lministration of the drug. These compositions can be prepared by mixin~ the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and while therefore melt in the rectum to release the drug, for example, cocoa butter and polyethylene glycols.
When the compounds of formula I are utilized in vivo, dosage levels on the order of from about 0.2 mg to about 300 mg, preferably from about 10 mg to about 100 mg, per kilogram of body weight per day are useful.
3 Aqueous suspensions usually contain the active materials in admixture with appropriate excipients. Such excipients are suspending agents, for exarnple, sodium carboxymethylcellulose, methylcellulose, hydroxypropymethylcellulose, sodium ~lin~te, polyvinylpyrolidone, gum WO 95/10286 - PCT/US94/11497 ~
~13~ s tragacanth and gum acacia; dispersing or wetting agents which may be a naturally-occuring phosphatide, for example, lecithin; a condensation product of an aLkylene oxide with a fatty oxide, for example, polyoxyethylene stearate; a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethylene-5 oxycetanol; a condensation product of ethylene oxide with a partial esterderived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate; or a condensation product of ethylene oxide with a partial ester derived from fatty acids and hexitol anhydrides, for example, polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example, ethyl, n-propyl, or p-hydroxybenzoate; one or more coloring agents; one or more flavoring agents; and one or more sweetening agents such as sucrose or saccharin.
Oily suspension may be form~ ted by suspending the active ingredients in a vegetable oil, for example, arachis oil, olive oil, sesame 15 oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspension may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an 20 antioxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in ~lmixtllre with a dispersing or wetting agent, suspending and one or more preservatives. Suitable dispersing or wetting agents and 25 suspending agents are exemplified by those already mentioned above.
Additional excipients, for example, sweetening, flavoring and coloring agents, may also be present.
~ or topical ~(lmini~tration, the ph~rm~ceutical composition may be in the form of a salve, cream, ointment, spray, powder or the like.
3 Standard pharrnaceutical carriers for such compositions may be used.
Preferably, compositions for topical ~lmini~tration will contain 0.05-5%
of the active ingredient.
A typical cream form~ tion may contain the following:

~ WO95/10286 2173~ ` ~ PCT/US91/11497 In~redient Parts by Wei~ht Water/glycol mixture 50-99 (15% or more glycol) Fatty alcohol 1-20 Non-ionic surfactant 0-10 Mineral oil 0-10 Typical ph~rm~ceutical 0-5 adjuvants Active ingredient 0.05-5 A typical ointment formulation may contain the following:

Ingredient Parts by Weight White petrolatum 40-99 Mineral oil 5-20 Glycol solvent 1-15 Surfactant 0-10 Stabilizer 0-10 Active Ingredient 0.05-5 The compounds of this invention may be combined with other known anti-infl~mm~tory/immlmosuppressive agents such as steroids or non-steroidal anti-infl~mm~tory agents (NSAID) in the pharmaceutical compositions and methods described herein.
Dosage levels of the order from 0.2 mg to 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (10 mg to 7 gms per patient per day). For example, infl~mm~tion is effectively treated and anti-pyretic and analgesic activity manifested by the ~(lmini~tration from about 0.5 to 50 mg of the 3 o compound per kilogram of body weight per day (25 mg to 3.5 gms per patient per day). Preferably, a dosage of from about 2 mg to about 20 mg per kilogram of body weight per day is used to produce effective results (50 mg to 1 gm per patient per day).

i f ~

WO 95/10286 ~ 3 4~G PCT/US94/11497 Similarly with regard to treatment of certain forms of cancer, a-lmini~tration is in an amount between about 0.1 mg/kg of body weight to about 20 mg/kg of body weight per day, preferably of between 0.5 mg/kg of body weight to about 10 mg/kg of body weight per day.
The amount of active ingredient that may be combined with 5 the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of ~lmini~tration. For example, a formulation intended for the oral ~lmini~tration of humans may contain from 5 mg to 5 gm of active agent compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to 95 percent of the total composition. Dosage unit forms will generally contain between about 25 mg to about 500 mg of active ingredient.
It will be understood, however, that the specific dose level for any particular patient will noImally be determined by the prescribing l 5 physician and will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of ~lmini~tration, route of ~lmini~tration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

EXPERIMENTAL PROCEDURES

l-Hydroxy-1-(3-(3-chlorophenoxy)phenyl)methylphosphonic acid Step A: 3-(3-Chlorophenoxy)benzaldehyde A mixtllre of 3-bromobenzaldehyde (6.0 g), 3-Chlorophenol (4.8 g), sodium hydride (1.3 g of an 80% suspension in oil) and cuprous 3 cloride (1 g) was heated under reflux in pyridine (125 ml) under an atmosphere of nitrogen gas for 16 hours. The mixture was cooled, diluted with water, acidified with hydrochloric acid and extracted with diethyl ether. The etheral extracts were dried (Na2S04), concentrated WO95/10286 73~S~ PCT/US9~/11497 , J
- 19- ~ ' :
and puri~led by chromatography on silica gel eluting with ethyl acetate-petroleum ether (1 :9) to give the title compound as a colourless oil.

Step B: Dimethyl 1 -Hydroxy- 1 -(3 -(3 -chlorophenoxy)phenyl)methyl -phosphonate The foregoing aldehyde (1.5 g) was stirred for 24 hours in a mixture of triethyl~mine (0.9 ml) and dimethyl phosphite (0.89 ml). The mixture was evaporated under reduced pressure and crystallised from ethyl acetate to give the tile compound as a white solid.

o Step C: 1-Hydroxy-1-(3-(3-chlorophenoxy)phenyl)methyl-phosphonic acid The phosphonate ester of part b) (0.5 g) was treated with trimethylsilyl bromide (0.96 ml) for 16 hours after which time the mixture was evaporated under reduced pressure and the residue dissolved in methanol (5 ml) for 10 minlltes. The solution was concentrated and the residue dissolved in water and freeze dried to give the title compound as a white solid. lH NMR (360 MHz, d6 DMSO) ~ 4.70 (lH, d, J=14Hz), 6.91-6.97 (2H, m), 7.02 (lH, t, J=2.2Hz), 7.13 (lH, d, J=1.5Hz),7.17 (lH, d, J=6.2Hz),7.23 (lH, d, J=7.4Hz),7.32-7.40 (2H, 20 m).

Examples 2-7 were prepared by the method of Fx~mple 1 using the appropriately substituted phenol.

l-Hydroxy-1-(3-phenoxyphenyl)methylphosphonic acid lH NMR (360 MHz, D2O) ~ 4.93 (lH, d, J=12.8Hz),7.00 (lH, d, J-8.1Hz),7.08 (2H, d, J=7.8Hz),7.14 (lH, d, J=1.9Hz),7.20 (lH, 30 t, J=7.6Hz),7.25 (lH, d, J=7.6Hz),7.38-7.45 (3H, m).

WO 95/10286 PCT/US9~/11497 ~

l-Hydroxy-1-(3-(3.4-dichlorophenoxy)phenvl)methylphosphonic acid lH NMR (360 MHz, d6 DMSO) ~ 4.70 (lH, d, J=14.3Hz), 6.94-7.01 (2H, m), 7.12 (lH, d, J=1.7Hz), 7.23-7.25 (2H, m), 7.35 (lH, t, J=7.8Hz), 7.61 (1 H, d, J=8.9Hz).

l-Hydroxy-1-(3-(4-chlorophenoxv)phenyl)methylphosphonic acid H NMR (360 MHz, d6 DMSO) o 4.67 (lH, d, J=14.3Hz), 6.88 (lH, d, J=7.0Hz), 7.00-7.02 (2H, dd, J=2.1 and 8.9Hz), 7.09 (lH, s), 7.19 (lH, d, J=7.0Hz), 7.31 (lH, t, J=7.0Hz), 7.39-7.42 (2H, dd, J=2.1 and 8.9Hz).

l-Hydroxy-1-(3-(2-chlorophenoxy)phenyl)methylphosphonic acid lH NMR (360 MHz, d6 DMSO) ~ 4.68 (lH, d, J=14.3Hz), 6.79 (lH, d, J=8.0Hz), 7.03-7.06 (2H, m), 7.17-7.21 (2H, m), 7.27-7.36 (2H, m), 7.58 (lH, dd, J=8.0 and 1.6Hz).

l-Hydroxy-1-(3-(4-methoxyphenoxy)phenyl)methylphosphonic acid lH NMR (360 MHz, d6 DMSO) ~ 3.74 (3H, s), 4.65 (lH, d, J=14.2Hz), 6.76 (lH, d, J=7.8Hz), 6.93-7.02 (5H, m), 7.09 (lH, d, J=7.8Hz), 7.23 (lH, t, J=7.8Hz).

WO 95/10286 ~ C, PCT/US94/11497 E~AMPLE 7 l-Hydroxy-1-(3-(4-methylphenoxy)phenyl)methvlphosphonic acid - 1H NMR (360 MHz, d6 DMSO) ~ 2.28 (3H, s), 4.65 (lH, d, J=14.2Hz), 6.80 (lH, d, J=8Hz), 6.90 (2H, d, J=9Hz), 7.04 (lH, d, J=l.SHz), 7.12 (lH, d, J=8Hz), 7.17 (2H, d, J=9Hz), 7.27 (lH, t, J=8Hz).

1-Hydroxy-1-(3-(4-hydroxyphenoxy)phenyl)methylphosphonic acid oDimethyl 1 -Hydroxy-1 -(3-(4-methoxyphenoxy)phenyl)-methylphosphonate was prepared by the method of Example la) and lb) using 4-methoxyphenol. A solution of this compound (150 mg) in dry dichloromethane (10 ml) at -78C under an atmosphere of nitrogen was treated with boron tribromide (4 ml of a lM solution in 15 dichloromethane). T~he solution was allowed to warm to 20C then concentrated under reduced pressure. The residue was treated with trimethylsilyl bromide (1.2 ml) for 3 hours then concentrated and dissolved in met~anol. After 10 mimltes the solution was concentrated under reduced pressure. The residue was dissolved in water and freeze 20 dried to give the title compound as a white solid. lH N~R (360 MHz, D2O) o 4.93 (lH, d, J=12.6Hz), 6.88-7.02 (SH, m), 7.07 (lH, d, J=1.8Hz), 7.19 (lH, d, J=7.5Hz), 7.37 (lH, t, J=7.5Hz).

1-Hydroxy-1-(4-(3-chlorophenoxy)phenyl)methylphosphonic acid Step A: 4-(3-Chlorophenoxy)benzaldehyde 3-Chlorophenol (1.9 g) was stirred with 4-fluoro-30 benzaldehyde (1.8 g) and sodium hydride (0.46 g of an 80% suspension in oil) in dimethylform~mide (50 ml) at 110C under an atmosphere of nitrogen for 16 hours. The solution was diluted with water and extracted with diethyl ether. The ethereal extract was dried (Na2S04), WO 95/10286 '~ ` ~ ; PCT/US94/11497 ~

concentrated and purified by chromatography on silica gel eluting with ethyl acetate-petroleum ether (1 :9) to give the title compound as a colourless oil.

Step B: 1-Hydroxy-1-(4-(3-chlorophenoxy)phenyl)methyl-phosphonic acid This was prepared from 4-(3-Chlorophenoxy)benzaldehyde using the methods of Example lb) and lc). lH NMR (360 MHz, d6 DMSO) ~ 4.69 (lH, d, J=13.6Hz), 6.92-7.02 (4H, m), 7.15-7.18 (lH, m), 7.37-7.46 (3H, m).

1-Hydroxy-1-(4-(3-ethylphenoxy)phenyl)methylphosphonic acid Prepared by the method of Example 9 using 3-e~ylphenol.
lH NMR (360 MHz, d4 MeOH/CDC13) ~ 1.21 (3H, t, J=7.6Hz), 2.61 (2H, q, J=7.6Hz), 4.90 (lH, d, J=11.7Hz), 6.78 (lH, dd, J=2.1 and 8.0Hz), 6.84 (lH, s), 6.94 (lH, d, J= 7.0Hz), 6.97 (2H, d, J=8.6Hz), 7.22 (lH, t, J=7.8Hz), 7.46 (2H, dd, J=1.8 and 8.6Hz).

1-Hvdroxy-l-f4-(3-methoxyphenoxy)phenyl)methylphosphonic acid Prepared by the method of Fx~mI)le 9 using 3-methoxy-phenol and converted to the bis-cyclohexylammonium salt by treating 25 . with cyclohexyl~mine in a mixture of methanol and ethylacetate, then ltering and drying. 1H NMR (360 MHz, D20) ~ 1.14-1.40 (lOH, m), 1.63-1.67 (2H, m), 1.77-1.81 (4H, m), 1.96-1.99 (4H, m), 3.09-3.16 (lH, m), 3.80 (3H, s), 4.71 (lH, d, J=11.4Hz), 6.71-6.73 (2H, m), 6.78-6.81 (lH, m), 7.06 (2H, d, J=8.6Hz), 7.32-7.37 (lH, m), 7.48 (2H, d, 30 J=8.6Hz).

WO95/10286 73~S~ PCT~S9~/11497 Assay Protocol for Evaluating Inhibition of Phospholipase C~
General. Competent DHSoc cells (subcloning efficiency) were obtained from GIBCO/BRL (Gaithersburg, MD). Competent BL21(DE3) cells were purchased from Novagen (Madison, VVI). PCR
mutagenesis was carried out according to literature protocol (Higuchi, 1990). ~he general cloning vector, pBSII(S/K)+, was from Stratagene (La Jolla, CA). pTST was constructed as described (Eisenberg et al., 1990). DNA sequencing was performed at each sequence modification on the relevant portion of the gene using the dideoxy chain terrnin~tion method (Sanger et al., 1977) to verify the change to the wild type cDNA.
Standard DNA manipulations were carried out as described (Sambrook et al., 1989).
Oligonucleotides. Synthetic deoxyribonucleotides were obtained from Midland Certified Reagent Co. (Midland, TX). The sequences of the oligonucleotides are (5' to 3'):

ATG-GCG-GGC-GCC-GCG-TCC (SEQ.ID.NO.: 4) 02 CTG-CTT-CCG-GAG-CCA-CCT-CTC (SEQ.ID.NO.: 5) 03 TC-GCC-ATT-CGT-CCT-GAG-GGC (SEQ.ID.NO.: 6) GCG-GTT-GTC-TCC-ATT-GAC-CCG-AGT-TCG-TCG
(SEQ.ID.NO.: 7) (SEQ.ID.NO.: 8) (SEQ.ID.NO.: 9) 3 Subcloning of the PLC~ Coding Sequence. Rat brain cDNA
is synthesized using rat brain poly(A)RNA as template by literature protocol (Sanbrook, J. et al., Molecular Cloning: A Laboratry Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). From WO 95/10286 13~56 ~ ~ PCT/US94/11497 ~

the published cDNA sequence of rat brain PLC yl (Suh, P.-G., Ryu, S. H., Moon, K. H., Suh, H. W., & Rhee, S. G. (1988) Proc. Natl. Acad. Sci.
USA 85:5419-5423), PCR primers, one which contains a BamHI
restriction site upstream of the sequence complementary to the 5' end of the PLC~l gene (primer 01), and the other one which contains a HindIII
5 site downsteam of the 3' end of the PLC~ gene (primer 04), were synthesized. Carrying out the PCR using these primers (01 and 04) with the above mentioned rat brain cDNA as template, a DNA fragment with the entire rat brain PLC~1 coding sequence, flanked by a 5' BamHI site and a 3' Hindm site, is generated. This BamHI-Hindm fragment 10 cont~ining the coding sequence of PLC~was subcloned into pBSII(S/K)+, generating pPLC1. The rest of the manipulations were accomplished using PCR to generate the appropriate DNA fragment. The S' end of the PLC y1 gene was altered to include a new BamHI site and asequence that would eventually translationally couple the expression of 15 PLC~l to the q)10 gene of the pT5T vector as described by Eisenberg, S.
P., Evans, R. J., Arend, W. P., Verderber, E., Brewer, M. T., ~nnllm, C.
H., & Thompson, R. C. (1990) Nature(London) 343:341-346.
Primers 01 and 02 were used to generate a 508 bp fragment (from template DNA pPLC1) cont~inin~; a 5' BamHI site and 3' EcoRI
2 site. This new restriction fragment was substituted for the analogous fragrnent in pPLC1 to produce pPLC2.
PCR-mediated mutagenesis with template pPLC2 and with primers 03-05 to generate one mllt~nt fragment and primers 04-06 to generate the second mutant fragment was used to restructure the 3' end, to 25 add DNA sequence encoding the epitope tag Glu-Glu-Phe (which is recognized by the monoclonal antibody YL1/2) (Kilmartin, J. V., Wright, B., & Milstein, C. (1982) J. Cell. Biol. 93:576-582), and to change the tandem AGG-AGG codons to CGG-CGG at amino acid positions 944-945 and 1279-1280. Tandem AGG codons are associated with poor 30 protein expression in E. coli (Bonekamp, F., & Jensen, K. (1988) Nucl.
AcidsRes. 17:3013-3024).
Using the isolated mllt~nt fragments from each of these PCRs together as template, the final PCR was carried out using primers ~ WO 95/10286 21 7 ~?e~ PCT/US94/11497 03-06 which yielded, after restriction enzyme digestion, a SphI and Hind~I fragment which was used to replace the analogous fragment in pPLC2. The resulting plasmid, pPLC3, contained the coding sequence of the PLC~l gene on a BamHI-Hind~ fragment with both the sequence encoding the Glu-Glu-Phe tag at the 3' end as well as the changed AGG
codons. Figure 3 shows the final cDNA sequence encoding the enzyme and its epitope tag. (SEQ.ID.NO.: 3) Finally, the BamHI-Hind~I
fragment from pPLC3 was transferred into the BamHI-Hina~I site of pT5T, generating pTST-PLC4. This construct was used to produce PLC~1 (cont~ining the Glu-Glu-Phe epitope tag at the C-terminus) tr~n~l~tionally coupled to the expression of plO protein in the pT5T
vector.
Expression and Pur~fication of PLC~y 1- To express PLC~1 the plasmid pT5T-PLC4 was transformed into E. coli BL21 (DE3). The transformed cells were grown in LB media cont~ining ampicillin (100 15 ,ug/ml) and tetracycline (12.5 ,ug/ml) at 20C until the optical density of the cultures at 550 nm was equal to 0.8. This transformed bacterial cell has been deposited with the ATCC and has been assigned the number ATCC 69421. Expression of PLC~l was then induced by addition of isopropyl ~-D-thiogalactopyranoside (0.5 mM final concentration) to the 20 cultures. After growing for another 6 hours the cells were harvested and PLCyl was purified as described below. PLC~was isolated from E. coli by resuspending a cell pellet in standard buffer, 50 mM Tris-Cl pH 8.0, 2 mM MgC12, 10 mM CaC12, 1 mM EGTA, 5 mM DTT, 5 mM
streptomycin sulfate, 1 mM PMSF, 2 ,ug/ml leupeptin, 2 ,ug/ml antipain, 5 10 ,ug/ml aprotinin (approxim~tely 5 g wet packed cells/10 ml buffer).
The resuspended cells were broken by sonication and the cell debris pelleted by centrifugation at 30,000 x g at 4C for 30 minlltes. The soluble fraction was applied at a flow rate of approximately 0.5 ml/min to a 2 ml column of the monoclonal antibody YL1/2 (4 mg antibody/ml 30 resin) coupled to cyanogen bromide activated Sepharose. The YL1/2 Sepharose column, which binds the epitope tag Glu-Glu-Phe had previously been equilibrated with standard buffer. After loading the protein onto the column, the column was washed with standard buffer WO 95/10286 ' " G PCTIUS9~/11497 ~

~ - 26 -(100 mls) PLC~ was eluted with 3 x 5ml 5 mM Asp-Phe dipeptide (Sigma) in standard buffer. The column was regenerated by washing with phosphate buffered saline (PBS) + 2 M NaCl and then stored in PBS
+ 0.02% NaN3 (wt./vol). The PLC~1 was obtained in >80% purity and in a 0.05 to 0.5% yield based on the total starting soluble E. coli protein. In 5 some cases the the PLC~1 was futher purified. This is not necessary for routine drug screening. To further purify the PLC~1 the protein eluted from the YL1/2 column was chromatographed by HPLC on a MonoQ
HR10-10 column (Pharmacia) where buffer A was standard buffer and buffer B was standard buffer + lM KCl. The column was run at 1 ml/min and the gradient was 0-30% B in 40 min., 30-50% B in 50 min., 50-100% "B" in 70 min. PLC~l eluted at approxim~tely 25-30% B.
Assay of purified PLC y l activity. Activity of the purified PLC yl was assayed at 30C. Reactions were never allowed to proceed to more than 10% completion based on the limiting substrate. A typical reaction contained the following: 50 mM HEPES pH7.5, 0.1%
Deoxycholate, 3 mM CaC12, 1 mM EGTA, 0.1 mM DlYr with phosphatidyl inositol (l-1000 ,uM) (PI) and 0.02 uCi [3H]-phosphatidyl inositol (PI) as substrate. The phospholipid components were dried under a gentle stream of nitrogen and resuspended in assay buffer. The substrate mix is then vortexed and sonicated (10 sec. with probe sonicator) to disperse the lipid and form micelles. After thermally preequilibrating the assay mixture in the absence of enzyme, ~e reaction was initi~ted by ~ckling PLC~yl. Reactions co~ 0.2 ml aliquots were termin~ted by addition of 1/4 volume 1 N HCl, S mM EGTA and transferred to an ice bath. The quenched reactions are then filtered through a Q sepharose (Ph~ cia) column. To prepare the Q sepharose column 1 ml of Q Sepharose slurry is added to a disposable plastic column. The resin is equilibrated by passing through 20 ml of 10 mM
NH4H2P04, pH 3.5. The quenched reaction, typically 200 ,ul, is applied 30 to the column and 3 ml of 10 mM NH4H2P04, pH 3.5 is added, the flow ~rough from this step is collected in a scintill~tion vial, mixed with 10 ml of scintill~tion fluid and counted in a Beckman LS3801 scintill~tion counter.

WO 95/10286 1 7~56 PCT/US9~/11497 Assay of inhibitory activity of the compounds of the invention. The inhibitory activity of the compounds of formula I against PLC~yl was assessed by including known concentrations of the compound of formula I in the assay mixture described above prior to the addition of the enzyme. The relative inhibitory concentrations calculated from the 5 assay are shown in Tables 1 and 2.

TABLE l (HO)2OP~ OH R4 R~ R5 Example No. R3 R4 R~ IC50 (~lM) l H -Cl H 10 2 H H H l90 3 H -Cl -Cl 56

4 H H -Cl 36 S -Cl H H 17 t ~ J'' ~ ~

WO 95/10286 ~ 4~ PCT/US91/11497 ~

HO~ ~ R4 Example No. R3 R4 R5 IC50 (~M) 9 H -Cl H 5 H -cH2cH3 H 37 H H H >300 20 Assay Protocol for Evaluating Inhibition of Phospholipase C-~ and PuriJ&cation of Phospholipase C-,(3 and ~from Bovine Brain PLC-,B are obtained as described by S. G. Rhee et al., Meth.
Enzym., 197:502-511 (1991). Specifically PLC-~ is purified from the cytosolic fraction of bovine brains, while PLC-,~ is mainly obtained from 25 the particulate fraction. However, since the particulate and cytosolic forms of PLC-,B are identical with respect to amino acid sequence, PLC-,~-cont~ining fractions pooled during the purification of the cytosolic PLC-~ are combined with the PLC-~B fraction from the particulate fraction. A total of 36 bovine brains are used for the purification.
3 Twelve brains are processed at one time.

Step 1: Separation of Cytosolic and Particulate Fractions ~ welve bovine brains are freshly obtained from a local slaughterhouse, and the cerebra (3.3 kg) are homogenized in a Waring WO 95/10286 ; ~1 73~S PCT/US94/11497 :
; .
- 29.-blender with 6.6 liters of buffer cont~ining 20 mM Tris-HC1, pH 7.4, 5 mM EGTA, 2 mM phenylmethysulfonyl fluoride (PMSF), and 0.1 mM
DTT. The homogenate is centrifuged for 30 min at 13,000 g at 4. Both the precipitate and supernatant are saved for Steps 2 and 3, respectively.

5 Step 2: Preparation of Extracts from Particulate Fractions The precipitate from Step 1 is resuspended in the same homogenization buffer (6.6 liters) and homogenized again to ensure complete breakage of cells. The homogenate is centrifuged for 30 min. at 13,000 g. The washed pellet is suspended in 2 M KC1 in homogenization buffer and stirred for 2 hr at 4. The suspension is then centrifuged for 90 min at 13,000 g. The supern~t~nt is brought to 60%
(NH4)2S04 saturation by ~lcling solid salt. This suspension is centrifuged for 30 min at 13,000 g, and the pellet is suspended in 500 ml of homogenization buffer; the suspension is dialyzed overnight against 15 the homogenization buffer. Dialyzed solution is centrifuged for 30 min at 13,000 g to remove insoluble particles, and the supern~t~nt, which is still very turbid, is kept at -20 to be combined with the dialyzed solutions from the two other identical preparations.

20 Step 3: Preparation of Cytosolic Extracts The supernatant from Step 1 is adjusted to pH 4.8 with 1 M
acetic acid. After 30 min at 4, precipitates are collected by centrifugation arl d dissolved in 1 liter of homogenization buffer.
Insoluble materials are pelleted by centrifuging for 30 min at 13,000 g, 25 and the turbid supern~t~nt is removed for Step 4.

Step 4: Ion-Exchange Chromatography on DEAE-Cellulose The supernatant from Step 3is applied to a DE-52 DEAE-cellulose (Wh~ n Biosystems, Maidstone, UK) column (8 x 40 cm), 3 0 which has been equilibrated with 20 mM Tris-HCl, pH 7.6, 1 mM EGTA, 0.1 mM DTT. The column is eluted with a 6-liter linear KC1 gradient from 0 to 225 mM KC1 in 50 mM Tris-HC1, pH 7.6, 1 mM EGTA, and 0.1 mM DTT. Three PLC activity peaks are eluted and the peak fractions

6 ; PCT/US9~/11497 ~
?J~3~6~

are pooled separately. The first peak, which contairis PLC-~, is further purified immediately in the next step. The second peak fractions cont~inin~ PLC-,B are concentrated to about 100 ml and combined with the extracts of particulate fractions from Step 2. The third peak fractions cont~ining PLC-y are discarded.

Purification of PLC-~

Step 5: Hçparin-Agarose Chromato~raphy of PLC-~
The PLC-o fractions pooled from the previous step (750 ml) are directly applied to a heparin-agarose column (5 x 15 cm) equilibrated with 20 mM HEPES, pH 7.0, 0.1 mM DTT, and 1 mM EGTA. The column is eluted with a 1.8-liter linear gradient of NaCl from 100 to 700 mM NaCl in equilibration buffer. The peak fractions (240 ml) are pooled, concentrated to approximately 10 ml in an Amicon (Danvers, MA) filtration apparatus, and stored frozen to be combined with concentrated fractions of PLC-o from two other identical preparations.

Step 6: Reversed-Phase Chromatography of PLC-~ on TSK
Phenvl-5-PW
Solid KCl is added to the combined concentrated fractions (35 ml) from Step 5 to give a final concentration of 3 M, and the mixtures are centrifuged to remove denatured proteins. The supern~t~nt~ are applied at a flow rate of 5.0 ml/min to a high-performance liquid chromatography (HPLC) preparative TSK phenyl-5-PW column (21.5 x 25 150 mm; Bio-Rad, Richmond, CA) equilibrated with 20 mM HEPES, pH

7.0, 3 M KCl, lmM EGTA, and 0.1 mM Dl~. Elution is continued at 5.0 ml/min with a decreasing KCl gradient from 3 to 1.2 M KCl for 10 min and with a decreasing KCl gradient from 1.2 to 0 M KCl for 20 min. Fractions (25 ml) cont~ining PLC activity are pooled and washed in 3 an Amicon filtration apparatus with 20 mM MOPS buffer, pH 5.7, 0.1 mM DTT, 1 mM EGTA, and finally concentrated to about 10 ml.

WO 95/10286 ~l ;~s PCT/US911/11497 Step 7: Ion-Exchange Chromatography of PLC-o on a Mono S
Column The washed protein solution (~10 ml) from Step 6 is applied at a flow rate of 1.0 ml/min to a Mono S colurnn (70 x 6 mm, Ph~ cia, Piscataway, NJ) equilibrated with 20 mM MOPS, pH 5.7, 0.1 mM DTT, 5 and 1 mM EGTA. Elution is continued at 1.0 mlJmin with a NaCl gradient from 0 to 300 mM NaCl for 20 min and from 300 mM to 1 M
for 10 min. Peak fractions (1.2 ml) are collected manually, diluted with 2 ml of 20 mM HEPES (ph 7.0), concentrated in a Centricon micro-concentrator (Amicon) to approximately 0.5 ml, separated into aliquots, and stored at -20. A total of 0.3-0.6 mg of homogeneous PLC-o is obtained, with a yield of 2-4%.

Purification of PLC-,~

15 Step 8: Ion-Exchange Chromatography of PLC-,~ on DEAE-Cellulose Because of turbidity, the combined protein solution from Steps 2 and 3 cannot be chromatographed on a DEAE-cellulose column directly. Therefore, two stages of DEAE-cellulose chromatography, a 20 batch procedure followed by a column step, are employed. In the batch step, all of the combined proteins are absorbed on 2 liters of DEAE-cellulose equilibrated with 20 mM Tris-HC1, pH 7.6, cont~ining 5 mM
EGTA and 0.1 mM DTT. The DEAE-cellulose slurry is stirred and then collected in a 4-liter sintered glass (coarse) filter funnel. The DEAE-2 5 cellulose is washed with the equilibration buffer until it is free of turbidlipid materials and unbound protein. For the column procedure, the washed DEAE-cellulose is removed from the filter funnel, mixed with the equilibration buffer, and poured onto a colurnn already cont~ining a 10 cm high bed of equilibrated DEAE-cellulose (final dimension, 8 x 45 3 cm). The column is eluted at a flow rate of 8 ml/min with an 8-liter linear gradient from 0 to 300 mM KCl buffer cont~ining 50 mM Tris-HCl, pH 7.6, 1 mM EGTA, and 0.1 mM DTT. The activity peak is eluted WO95/10~8G ?~3~5 PCT159J/li~197 ~

at a KCl concentration of 110 mM. The peak fractions (600 ml) are pooled.

Step 9: Heparin-Agarose Chromatography of PLC-~
The pooled fraction from Step 8 (600 ml) is applied to a 5 heparin-agarose column (5 x 25 cm) equilibrated with 20 mM HEPES, pH 7.0, 100 mM NaCl, 0.1 mM DlYr, and 1 mM EGTA. The column is eluted with a linear gradient from 100 to 500 mM NaCl in 1.5 liters of equilibrium buffer. Peak fractions (310 ml) are pooled and concentrated on an Amicon filter to 27 ml.

Step 10: Reversed-Phase Chromatography of PLC-,~ on TSK
Phenyl-5-PW
Solid KCl is added to the concentrated fractions from Step 9 to give a concentration of 3 M, and the mixtures are centrifuged to 15 remove denatured proteins. The supem~t~nt~ are applied at a flow rate of S ml/min to an HPLC preparative phenyl-5-PW column (150 ~ 215 mm) equilibrated with 20 mM HEPES, pH 7.0, 3 M KCl, 1 mM EGTA, and 0.1 mM DTT. Elution is continued at 5 ml/min with a decreasing KCl gradient from 3 to 1.2 M for 15 min and with a decreasing gradient from 20 1.2 to 0 M for 20 min. Then the column is washed with a KCl-free buffer. Fractions cont~ining each of the two peaks of PLC activity (15 ml for fraction Ml and 13 ml for fraction M2) are collected separately. The pooled solutions are washed with a KCl-free 20 mM HEPES, pH 7.0, and are concentrated to 5 ml in an Amicon filter concentrating procedure.
25 Analysis on SDS-polyacrylamide gels indicates that fractions Ml and M2 contain 150-kDa (PLC-~l) and 140-kDa (PLC-,B2) forms of PLC, respect*ely. The two forms are immllnologically indistinguishable.
Whether PLC-~B2 is a proteolytic fragment of PLC-~l or a product of alternately spliced mRNA is not known. About 15 mg of PLC-~l and 8 3 mg of PLC-~2 are obtained.

Assay of inhibitory activity of the compounds of the invention. The inhibitory activity of the compounds of formula I against WO 95110286 ~' ' J ~ . PCT/US94/11497 21 734~
, ~, PLC~ was assessed by including known concentrations of the compound of formula I in an assay mixture similar to the assay mixture described in Example 12, but substituting PLC,B obtained as described above for PLC~
of Example 12, prior to the addition of the enzyme. The relativeinhibitory concentrations calculated from the assay are shown in Tables 3 5 and4.

(HO)20P ~OH R4 [~ R~ R5 Example No R3 R4 RS IC50 (~uM) 2 o 1 H -Cl H 24 2 H H H >300 3 H -Cl -Cl 110 -4 H H -Cl 65 S -Cl H H 38 25. 6 H H -OCH3 717

8 H H -OH 7 WO 95/10~8G , ~ } ~ ' PCT I S9J/11-197 ~

HO~ ~ R4 ExampleNo. R3 R4 RS IC50 (~lM)9 H -Cl H 9 H -cH2cH3 H 74 H H H >300 The inhibitory activity of the compounds of formula I
20 against PLC~ is assessed by including known concentrations of the compound of formula I in an assay mi~lur~ simil~r to ~e assay mixture described in Fx~mple 12, but sub~lilulhlg PLCo obtained as described above for PLC~ of Fx~mI~le 12, prior to ~e addition of the enzyme.

W O 95/10286 ~ PCTrUS94/11497 SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: GIBBS, JACKSON B.
KOBLAN, KENNETH S.
MACLEOD, ANGUS M.
MERCHANT, KEVIN J.
(ii) TITLE OF INVENTION: INHIBITORS OF PHOSPHOINOSITIDE-SPECIFIC
PHOSPHOLIPASE C
(iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: DAVID A. MUTHARD
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(A) NAME: MUTHARD, DAVID A.
(B) REGISTRATION NUMBER: 35,297 (C) REFERENCE/DOCKET NUMBER: 18938 (ix) TELECOMMUNICATION INFORMATION:
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W O 95/10286 PCTrUS94111497 ~
G

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GGAGAGCGGC CAGAGCTTTG CCAGGTGTCC CTTTCTGAGT TCCAGCAGTT C~ll~'ll~AG 780 TTT~l~l~l~l CCAAAGAGAA CA~l~l~l~G AACTCACAGC TGGATGCCGT GTGCCCAGAA 960 ACCATGAACA ACCCACTGTC TCACTATTGG A'l~'l'~'l"l~CT CGCATAATAC GTATCTGACT 1020 ATTAAGCATA AGAAGCTGGC TGAGGGCAGT GCCTATGAGG AGGTGCCTAC ~'l'~'l~l'GATG 1440 W 09StlO286 ~3-~G PCTtUS94/11~97 GG~lC~ll~C TAGTGCGAGA AAGTGAGACC TTCGTGGGGG ACTACACGCT ~l~lllll~G 1800 T~l~cT~l~L~l"l~A CAGATAACCT 'l~'l'~"l"l"l~AC T~ ~T~TATG ACCTCATCAC ACATTATCAG 1920 TCCTATGCCA l~l'~`'l"l"l'CCG GGCTGAGGGA AAGATCAAGC ACTGCCGAGT ACAGCAGGAA 2160 ~'l"l"l'~'l'ATG TGGAGGCCAA CCCTATGCCA ACTTTCAAGT GTGCAGTAAA AGCTCTCTTC 2400 CATCTGGATG AGAACAGCCC ACTGGGGGAC TTGCTGCGAG G~l~l"l'AGA TGTGCCAGCC 2640 TGCCAGATCG CCAll~C~lcC TGAGGGCAAA AACAACCGGC 'l'~'l"l'~'l'~'l"l' CTCCATCAGC 2700 GA~l~l~A AAAAGATCCG TGAAGTTGCC CAGACTGCAG ATGCCAGGCT TACTGAGGGG 2820 CGGCCTGTTC CCTTTGACGA AGAGAAGATT GGCACAGAAC GCG~ll~llA CCGGGACATG 2940 CTCCAGTACA ACCGGCTGCA G~'l'~l~lCGC ATCTACCCTA AGGGTCAGAG GCTGGACTCC 3060 TCCAATTATG ACC~l~l~CC CATGTGGATC TGTGGCAGCC AGCTTGTAGC TCTCAATTTT 3120 AGTCTCCGAG GTCTGGAGCC ~'l~'l~'l'CATT TGCATTGAGG TGCTGGGGGC CAGGCATCTG 3300 WO 95/1028G ~ G PCT/US94/11497 TACGACAGCA CCAAGCAGAA GACAGAGTTT GTAGTGGACA ATGGACTGAA CC~~ ~G 3420 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1290 amino acids (B~ TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ala Gly Ala Ala Ser Pro Cys Ala Asn Gly Cys Gly Pro Ser Ala Pro Ser Glu Ala Glu Val Leu His Leu Cys Arg Ser Leu Glu Val Gly Thr Val Met Thr Leu Phe Tyr Ser Lys Lys Ser Gln Arg Pro Glu Arg Lys Thr Phe Gln Val Lys Leu Glu Thr Arg Gln Ile Thr Trp Ser Arg Gly Ala Asp Lys Ile Glu Gly Ser Ile Asp Ile Arg Glu Ile Lys Glu Ile Arg Pro Gly Lys Thr Ser Arg Asp Phe Asp Arg Tyr Gln Glu Asp Pro Ala Phe Arg Pro Asp Gln Ser His Cys Phe Val Ile Leu Tyr Gly WO95/10286 . PCT/US9l/11497 7~s6 Met Glu Phe Arg Leu Lys Thr Leu Ser Leu Gln Ala Thr Ser Glu Asp Glu Val Asn Met Trp Ile Lys Gly Leu Thr Trp Leu Met Glu Asp Thr Leu Gln Ala Ala Thr Pro Leu Gln Ile Glu Arg Trp Leu Arg Lys Gln Phe Tyr Ser Val Asp Arg Asn Arg Glu Asp Arg Ile Ser Ala Lys Asp Leu Lys Asn Met Leu Ser Gln Val Asn Tyr Arg Val Pro Asn Met Arg Phe Leu Arg Glu Arg Leu Thr Asp Phe Glu Gln Arg Ser Gly Asp Ile Thr Tyr Gly Gln Phe Ala Gln Leu Tyr Arg Ser Leu Met Tyr Ser Ala Gln Lys Thr Met Asp Leu Pro Phe Leu Glu Thr Asn Thr Leu Arg Thr Gly Glu Arg Pro Glu Leu Cys Gln Val Ser Leu Ser Glu Phe Gln Gln Phe Leu Leu Glu Tyr Gln Gly Glu Leu Trp Ala Val Asp Arg Leu Gln Val Gln Glu Phe Met Leu Ser Phe Leu Arg Asp Pro Leu Arg Glu Ile Glu Glu Pro Tyr Phe Phe Leu Asp Glu Leu Val Thr Phe Leu Phe Ser Lys Glu Asn Ser Val Trp Asn Ser Gln Leu Asp Ala Val Cys Pro Glu Thr Met Asn Asn Pro Leu Ser His Tyr Trp Ile Ser Ser Ser His Asn Thr Tyr Leu Thr Gly Asp Gln Phe Ser Ser Glu Ser Ser Leu Glu Ala Tyr Ala Arg Cys Leu Arg Met Gly Cys Arg Cys Ile Glu Leu Asp Cys Trp Asp Gly Pro Asp Gly Met Pro Val Ile Tyr His Gly His Thr Leu Thr Thr Lys Ile Lys Phe Ser Asp Val Leu His Thr Ile Lys Glu His Ala Phe Val Ala Ser Glu Tyr Pro Val Ile Leu Ser Ile Glu Asp His W O 95/10286 ' PCTrUS9~/11497 ~
2~34~6 Cy5 Ser Ile Ala Gln Gln Arg Asn Met Ala Gln His Phe Arg Lys Val Leu Gly Asp Thr Leu Leu Thr Lys Pro Val Asp Ile Ala Ala Asp Gly Leu Pro Ser Pro Asn Gln Leu Lys Arg Lys Ile Leu Ile Lys His Lys Lys Leu Ala Glu Gly Ser Ala Tyr Glu Glu Val Pro Thr Ser Val Met Tyr Ser Glu Asn Asp Ile Ser Asn Ser Ile Lys Asn Gly Ile Leu Tyr Leu Glu Asp Pro Val Asn His Glu Trp Tyr Pro His Tyr Phe Val Leu Thr Ser Ser Lys Ile Tyr Tyr Ser Glu Glu Thr Ser Ser Asp Gln Gly Asn Glu Asp Glu Glu Glu Pro Lys Glu Ala Ser Gly Ser Thr Glu Leu His Ser Ser Glu Lys Trp Phe His Gly Lys Leu Gly Ala Gly Arg Asp Gly Arg His Ile Ala Glu Arg Leu Leu Thr Glu Tyr Cys Ile Glu Thr Gly Ala Pro Asp Gly Ser Phe Leu Val Arg Glu Ser Glu Thr Phe Val Gly Asp Tyr Thr Leu Ser Phe Trp Arg Asn Gly Lys Val Gln His Cys Arg Ile His Ser Arg Gln Asp Ala Gly Thr Pro Lys Phe Phe Leu Thr Asp Asn Leu Val Phe Asp Ser Leu Tyr Asp Leu Ile Thr His Tyr Gln Gln Val Pro Leu Arg Cys Asn Glu Phe Glu Met Arg Leu Ser Glu Pro Val Pro Gln Thr Asn Ala His Glu Ser Lys Glu Trp Tyr His Ala Ser Leu Thr Arg Ala Gln Ala Glu His Met Leu Met Arg Val Pro Arg Asp Gly Ala Phe Leu Val Arg Lys Arg Asn Glu Pro Asn Ser Tyr Ala Ile WO 95/10286 PCTrUS94/11497 .. ~;
.

Ser Phe Arg Ala Glu Gly Lys Ile Lys His Cys Arg Val Gln Gln Glu Gly Gln Thr Val Met Leu Gly Asn Ser Glu Phe Asp Ser Leu Val Asp Leu Ile Ser Tyr Tyr Glu Lys His Pro Leu Tyr Arg Lys Met Lys Leu Arg Tyr Pro Ile Asn Glu Glu Ala Leu Glu Lys Ile Gly Thr Ala Glu Pro Asp Tyr Gly Ala Leu Tyr Glu Gly Arg Asn Pro Gly Phe Tyr Val Glu Ala Asn Pro Met Pro Thr Phe Lys Cys Ala Val Lys Ala Leu Phe Asp Tyr Lys Ala Gln Arg Glu Asp Glu Leu Thr Phe Thr Lys Ser Ala Ile Ile Gln Asn Val Glu Lys Gln Asp Gly Gly Trp Trp Arg Gly Asp Tyr Gly Gly Lys Lys Gln Leu Trp Phe Pro Ser Asn Tyr Val Glu Glu Met Ile Asn Pro Ala Ile Leu Glu Pro Glu Arg Glu His Leu Asp Glu Asn Ser Pro Leu Gly Asp Leu Leu Arg Gly Val Leu Asp Val Pro Ala Cys Gln Ile Ala Ile Arg Pro Glu Gly Lys Asn Asn Arg Leu Phe Val Phe Ser Ile Ser Met Pro Ser Val Ala Gln Trp Ser Leu Asp Val Ala Ala Asp Ser Gln Glu Glu Leu Gln Asp Trp Val Lys Lys Ile Arg Glu Val Ala Gln Thr Ala Asp Ala Arg Leu Thr Glu Gly Lys Met Met Glu Arg Arg Lys Lys Ile Ala Leu Glu Leu Ser Glu Leu Val Val Tyr Cys Arg Pro Val Pro Phe Asp Glu Glu Lys Ile Gly Thr Glu Arg Ala Cys Tyr Arg Asp Met Ser Ser Phe Pro Glu Thr Lys Ala Glu Lys Tyr Val Asn Lys Ala Lys Gly Lys Ly~ Phe Leu Gln Tyr Asn Arg Leu Gln Leu WO 95/10286 PCT/US94/11497 ~

Ser Arg Ile Tyr Pro Lys Gly Gln Arg Leu Asp Ser Ser Asn Tyr Asp Pro Leu Pro Met Trp Ile Cys Gly Ser Gln Leu Val Ala Leu Asn Phe Gln Thr Pro Asp Lys Pro Met Gln Met Asn Gln Ala Leu Phe Met Ala 1045 lQ50 1055 ly Gly His Cys Gly Tyr Val Leu Gln Pro Ser Thr Met Arg Asp Glu Ala Phe Asp Pro Phe Asp Lys Ser Ser Leu Arg Gly Leu Glu Pro Cys Val Ile Cys Ile Glu Val Leu Gly Ala Arg His Leu Pro Lys Asn Gly Arg Gly Ile Val Cys Pro Phe Val Glu Ile Glu Val Ala Gly Ala Glu llQ5 1110 1115 1120 Tyr Asp Ser Thr Lys Gln Lys Thr Glu Phe Val Val Asp Asn Gly Leu sn Pro Val Trp Pro Ala Lys Pro Phe His Phe Gln Ile Ser Asn Pro Glu Phe Ala Phe Leu Arg Phe Val Val Tyr Glu Glu Asp Met Phe Ser Asp Gln Asn Phe Leu Ala Gln Ala Thr Phe Pro Val Lys Gly Leu Lys Thr Gly Tyr Arg Ala Val Pro Leu Lys Asn Asn Tyr Ser Glu Asp Leu Glu Leu Ala Ser Leu Leu Ile Lys Ile Asp Ile Phe Pro Ala Lys Glu sn Gly Asp Leu Ser Pro Phe Ser Gly Thr Ser Leu Arg Glu Arg Ala Ser Asp Ala Ser Ser Gln Leu Phe His Val Arg Ala Arg Glu Gly Ser Phe Glu Ala Arg Tyr Gln Gln Pro Phe Glu Asp Phe Arg Ile Ser Gln Glu His Leu Ala Asp His Phe Asp Ser Arg Glu Arg Arg Ala Pro Arg Arg Thr Arg Val Asn Gly Asp Asn Arg Leu ~ W O95/10286 PCTrUS94/11497 Z~

(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3893 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

ATGTACAGCG CCCAGAAGAC GATGGACCTT C~'l"l'~'l"l~G AAACCAACAC TTTGAGGACT 720 GGAGAGCGGC CAGAGCTTTG CCAG~l~lCC ~lll~lGAGT TCCAGCAGTT C~ll~ll~AG 780 TACCAGGGGG AG~l~l~GGC TGTCGACCGG CTTCAGGTGC AGGAATTTAT GCTCAGCTTC 840 'l"l"l'~'l~'l"l'~'l' CCAAAGAGAA CA~l~l~lGG AACTCACAGC TGGATGCCGT GTGCCCAGAA 960 ACCATGAACA ACCCACTGTC TCACTATTGG Al~l~llCCT CGCATAATAC GTATCTGACT 1020 l~lCG~l~CA TCGAGTTGGA CTGCTGGGAT GGGCCAGATG GGATGCCAGT CATTTACCAT 1140 GC~llC~lAG CCTCAGAGTA CCCTGTCATC CTGTCCATCG AGGACCACTG CAGCATTGCC 1260 W 095/10286 ~ 3 ~ 5 G PCTrUS94/11497 ATTAAGCATA AGAAGCTGGC TGAGGGCAGT GCCTATGAGG AGGTGCCTAC ~l~l~l~ATG 1440 GGCTCCTTCC TAGTGCGAGA AAGTGAGACC TTCGTGGGGG ACTACACGCT ~l~'l"l"l"l"l'GG 1800 CGGAATGGGA AAGTCC~GCA CTGCCGTATC CACTCCCGGC AGGATGCTGG GACTCCTAAG 1860 GA CAGATAACCT ~ l~AC '~ ATG ACCTCATCAC ACATTATCAG 1920 TCCTATGCCA l~l~lllCCG GGCTGAGGGA AAGATCAAGC ACTGCCGAGT ACAGCAGGAA 2160 G~l~l~l~lATG TGGAGGCCAA CCCTATGCCA ACTTTCAAGT GTGCAGTAAA AGCTCTCTTC 2400 TGCCAGATCG CCATTCGTCC TGAGGGCAAA AACAACCGGC 'l'~'l"l'C~'l'~'l"l' CTCCATCAGC 2700 CGGCCTGTTC CCTTTGACGA AGAGAAGATT GGCACAGAAC GCG~'l"l~'l"l'A CCGGGACATG 2940 ~ W 095/10286 ~ 2173~SG

CAGACCCCAG ACAAGCCTAT GCAGATGAAC CAGGCCCTCT TCATGGCTGG TGGACACTGT 3l80 AGTCTCCGAG GTCTGGAGCC ~l~l~l~ATT TGCATTGAGG TGCTGGGGGC CAGGCATCTG 3300 CCGAAGAATG GCCGGGGTAT '~ ~'l~'l'~CT TTCGTGGAGA TTGAAGTGGC TGGGGCAGAG 3360 TACGACAGCA CCAAGCAGAA GACAGAGTTT GTAGTGGACA ATGGACTGAA CC~l~l~l~G 3420 CCTGCAAAGC CCTTCCACTT CCAGATCAGT AACCCAGAGT TTGCCTTTCT GCG~lll~TG 3480 AGCCCTTTCA GTGGTACATC CCTAAGGGAA CGGGCCTCAG ATGCCTCCAG CCA~l~llC 3720 (2~ INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CCCGGGCATA TGGATCCATT GGAGGATGAT TAAATGGCGG GCGCCGCGTC C 5l (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear WO 95/10286 ~ ~'' G PCT/US9~/11497 (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

WO95/10286 ' i ~ l43~56 PCTIUS9.1111497 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: slngle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Claims (16)

WHAT IS CLAIMED IS:

1. A compound of structural formula I:

I

or a pharmaceutically acceptable salt or ester thereof, wherein:
R1 and R2 are independently selected from:
a) hydrogen; and b) provided that if R1 is hydrogen, R2 is substituent b) and if R2 is hydrogen, R1 is substituent b); and R3, R4, R5 and R6 are independently selected from the group consisting of:

a) hydrogen;
b) halogen;

c) C1-C4-alkyl;
d) C1-C4-alkoxy; and e) hydroxy, provided that at least one of R3, R4, R5 and R6 is a substituent other than hydrogen and R3 and R4 are not simultaneously methyl.

2. The compound according to Claim 1 which has the formula:

wherein:

H H -Cl H -Cl H
-Cl H H

H H -OH or H -Cl -Cl.

3. The compound according to Claim 1 which has the formula:

wherein:

H -Cl H
H -OCH3 H or H -CH2CH3 H.

4. A pharmaceutical composition comprising a therapeutically effective amount of a compound of Claim 1 and a pharmaceutically acceptable carrier.

5. A method of inhibiting phospholipase C in a mammal in need of inhibitory treatment comprising the administration to the mammal in need of such treatment a therapeutically effective amount of a compound of the structural formula I:

or a pharmaceutically acceptable salt or ester thereof, wherein:
R1 and R2 are independently selected from:
a) hydrogen; and b) provided that if R1 is hydrogen, R2 is substituent b) and if R2 is hydrogen, R1 is substituent b); and R3, R4, R5 and R6 are independently selected from the group consisting of:

a) hydrogen;
b) halogen;
c) C1-C4-alkyl;
d) C1-C4-alkoxy; and e) hydroxy, provided that at least one of R3, R4, R5 and R6 is a substituent other than hydrogen; or R3 and R4 or R4 and R5 are combined to form a - CH2CH2CH2H2 -diradical.

6. A method of inhibiting phospholipase C according to Claim S wherein the compound is selected from the following compounds:

wherein:

H H -Cl H -Cl H
-Cl H H

H H -OH and H -Cl -Cl.

7. A method of inhibiting phospholipase C according to Claim 5 wherein the compound is selected from the following compounds:

wherein:

H -Cl H

H -CH2CH3 H and -CH3 -CH3 H.

8. A method of inhibiting phospholipase C according to Claim 5 wherein the compound is selected from the following compounds:

and

9. A method of inhibiting phospholipase C in a mammal in need of inhibitory treatment comprising the administration to the mammal in need of such treatment a therapeutically effetive amount of the compound according to Claim 1.

10. A method of treating cancer in a mammal in need of such treatment comprising the administration to the mammal in need of such treatment a therapeutically effective amount of a compound of the structural formula I:

or a pharmaceutically acceptable salt or ester thereof, wherein:
R1 and R2 are independently selected from:
a) hydrogen; and b) provided that if R1 is hydrogen, R2 is substituent b) and if R2 is hydrogen, R1 is substituent b); and R3, R4, R5 and R6 are independently selected from the group consisting of :

a) hydrogen;
b) halogen;
c) C1-C4-alkyl;
d) C1-C4-alkoxy; and e) hydroxy, provided that at least one of R3, R4, R5 and R6 is a substituent other than hydrogen; or R3 and R4 or R4 and R5 are combined to form a - CH2CH2CH2CH2 -diradical.

11. The method of treating cancer in a mammal according to Claim 10 wherein the form of cancer that is treated is breast cancer.

12. A method of treating cancer in a mammal in need of such treatment comprising the administration to the mammal in need of such treatment a therapeutically effective amount of the compound according to Claim 1.

13. A method of treating inflammation in a mammal in need of such treatment comprising the administration to the mammal in need of such treatment a therapeutically effective amount of a compound of the structural formula I:

I

or a pharmaceutically acceptable salt or ester thereof, wherein:
R1 and R2 are independently selected from:
a) hydrogen, and b) ;

provided that if R1 is hydrogen, R2 is substituent b) and if R2 is hydrogen, R1 is substituent b); and R3, R4, R5 and R6 are independently selected from the group consisting of:

a) hydrogen;
b) halogen;
c) C1-C4-alkyl;
d) C1-C4-alkoxy; and e) hydroxy, provided that at least one of R3, R4, R5 and R6 is a substituent other than hydrogen; or R3 and R4 or R4 and R5 are combined to form a -CH2CH2CH2CH2-diradical.

14. A method of treating inflammation in a mammal in need of such treatment comprising the administration to the mammal in need of such treatment a therapeutically effective amount of the compound according to Claim 1.

15. A method of treating benign epidermal hyperplasia in a mammal in need of such treatment comprising the administration to the mammal in need of such treatment a therapeutically effective amount of a compound of the structural formula I:

I

or a pharmaceutically acceptable salt or ester thereof, wherein:
R1 and R2 are independently selected from:
a) hydrogen; and b) ;
provided that if R1 is hydrogen, R2 is substituent b) and if R2 is hydrogen, R1 is substituent b); and R3, R4, R5 and R6 are independently selected from the group consisting of:

a) hydrogen;
b) halogen;
c) C1-C4-alkyl;
d) C1-C4-alkoxy; and e) hydroxy, provided that at least one of R3, R4, R5 and R6 is a substituent other than hydrogen; or R3 and R4 or R4 and R5 are combined to form a - CH2CH2CH2CH2 -diradical.

16. A method of treating benign epidermal hyperplasia in a mammal in need of such treatment comprising the administration to the mammal in need of such treatment a therapeutically effective amount of the compound according to Claim 1.